JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org.. ABSTRACTDuring bouts of torpor, mitochondrial metabolism is known to be suppressed in the liver and skeletal muscle of hibernating mammals. This suppression is rapidly reversed during interbout euthermic (IBE) phases, when whole-animal metabolic rate and body temperature (T b ) return spontaneously to euthermic levels. Such mitochondrial suppression may contribute significantly to energy savings, but the capacity of other tissues to suppress mitochondrial metabolism remains unclear. In this study we compared the metabolism of mitochondria from brain cortex and left ventricular cardiac muscle between animals sampled while torpid (stable T b near 5ЊC) and in IBE (stable T b near 37ЊC). Instead of isolating mitochondria using the traditional methods of homogenization and centrifugation, we permeabilized tissue slices with saponin, allowing energetic substrates and inhibitors to access mitochondria. No significant differences in state 3 or state 4 respiration were observed between torpor and IBE in either tissue. In general, succinate produced the highest oxidation rates followed by pyruvate and then glutamate, palmitoyl carnitine, and b-hydroxybutyrate. These findings suggest that there is no suppression of mitochondrial metabolism or change in substrate preference in these two tissues despite the large changes in whole-animal metabolism seen between torpor and IBE.

Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of CDKs are often drivers of cancer pathogenesis. Cyclin-dependent kinase 4 and 6 (CDK4)/(CDK6) are critical mediators of cellular transition into S phase and important for the initiation, growth, and survival of many cancers. Activated CDK4/CDK6 complexes phosphorylate Rb1, reduce their binding affinities and release Rb1-containing transcription repressor complexes from E2F transcription factors, resulting in activation of E2F controlled cell cycle genes and progression of the cell cycle. At present three CDK4/CDK6 inhibitors are approved for the treatment of ER+/HER2- breast cancer, and are being explored in other cancer indications as well. Previously we described a novel brain penetrant CDK4/CDK6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/CDK6 and >2000-fold selectivity against CDK1, CDK2 and CDK9. PRT3645 inhibits cellular phosphorylation of Rb and exhibits a protein binding-adjusted cellular IC50 of <300 nM. PRT3645 exhibits favorable in-vitro safety pharmacology and ADME properties, including increased brain penetration, and demonstrates oral bioavailability across rodents, dog, and non-human primates. In addition to robust monotherapy activity observed in preclinical models of ER+/HER2- breast cancer, we explored the activity of PRT3645 in other tumor types as well as in combination with other targeted therapies. In NSCLC, PRT3645 treatment resulted in significant inhibition of cell lines that harbor activation of the RAS/MEK/ERK pathway in proliferation assays and demonstrated comparable high synergy scores when combined with clinically approved covalent KRAS G12C inhibitors. In-vivo, oral PRT3645 was well tolerated and induced anti-tumor efficacy in two KRAS G12C mutant xenograft models that harbor the CDKN2A (p16) deletion. Anti-tumor efficacy was further improved when PRT3645 was combined with KRAS/MEK inhibitors in xenograft models and the combination therapy was well tolerated. In addition, we explored combinations of PRT3645 with a brain penetrant receptor tyrosine kinase inhibitor (TKI), an approved treatment for patients with advanced HER2+ breast cancer, including patients with brain metastases. In a HER2+ orthotopic human breast cancer brain metastasis model, PRT3645 was highly efficacious in combination with HER2 kinase inhibition and enhanced median survival significantly. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species and brain penetrance. In preclinical studies, PRT3645 was highly efficacious when combined with KRAS/MEK inhibitors, and with a brain penetrant HER2 receptor TKI, both in-vitro and in-vivo. PRT3645 has advanced into Phase 1 clinical trials (NCT05538572). Citation Format: Yue Zou, Srijita Dhar, Kirsten Gallagher, Andrew Buesking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Katarina Rohlfing, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Jing Ni, Jean Zhao, Bruce Ruggeri, Andrew Combs, Kris Vaddi, Sandy Geeganage, Ashish Juvekar, Sang Hyun Lee, Peggy Scherle. The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5973.

Cell cycle deregulation is a hallmark of cancer and CDK inhibitors, specifically inhibiting CDK4/6 and blocking cells transition from the G1 to the S phase of the cell cycle are the first and only class of highly specific CDK inhibitors approved for cancer treatment to date. CDK4/6 inhibitors have transformed the treatment paradigm of estrogen receptor-positive (ER+), HER2- breast cancer with three CDK4/6 inhibitors currently FDA approved. Brain metastasis commonly arises in patients with breast, lung, melanoma and other cancer types, is associated with poor survival outcomes and poses distinct challenges in clinical management. Due to advances in imaging technologies, the detection of brain metastases is increasing and there is a dearth of novel therapies to combat brain metastatic cancers and impact patient survival. Here, we describe a novel brain penetrant CDK4/6 inhibitor, PRT3645 exhibiting single digit nanomolar biochemical potency against CDK 4/6 and >2000-fold selectivity against other CDK family members (CDK1, CDK2, and CDK9). In cellular assays, PRT3645 inhibits cellular phosphorylation of RB with low nanomolar activity. Consistent with this, PRT3645 treatment resulted in concentration-dependent inhibition of cell proliferation in glioblastoma (GBM) cell lines and in HER2- and HER2+ breast cancer lines (EC50 values < 125 nM). Furthermore, PRT3645 demonstrated additive in vitro activity with fulvestrant or tucatinib in ER+ and HER2+ breast cancer lines. PRT3645 exhibits favorable in vitro safety pharmacology and ADME profiles, including brain exposure in rodents at steady state, and demonstrates oral bioavailability across rodents, dog and nonhuman primates. In vivo, oral PRT3645 was well tolerated and highly efficacious in a dose-dependent manner in subcutaneous xenograft models of GBM and breast cancer and in orthotopic human breast cancer brain metastasis (BCBM) and GBM models in mice as a monotherapy. PRT3645 showed tumor regression as single agent in the MCF7 ER+ breast cancer model and a combinatorial benefit with the estrogen receptor blocker, fulvestrant. In a HER2+ BT474-luc orthotopic model, similarly efficacious single agent activity of PRT3645 was achieved, as well as a significant combinatorial benefit on tumor growth and median survival when administered with the brain penetrant HER2 kinase inhibitor, tucatinib. PRT3645 was highly efficacious in a U87-luc GBM orthotopic model and demonstrated enhanced median survival benefit when combined with an orally active brain penetrant PRMT5 inhibitor. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species, brain penetrance and favorable tissue distribution relative to brain exposure, and currently has advanced into IND-enabling preclinical studies. Citation Format: Ashish Juvekar, Yang Zhang, Andrew Buesking, Min Wang, Dave Rominger, Joseph Rager, Stefan Ruepp, Kirsten Gallagher, Yue Zou, Miles Cowart, Xiaowei Wu, Sarah Pawley, Ryan Holmes, William Gowen-MacDonald, Kris Vaddi, Andrew Combs, Bruce Ruggeri, Peggy Scherle. Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2300.